AbstractBackgroundVaricocoele‐associated stressors, such as hypoxia and heat, can damage cell function and viability, and some exosomal biomarkers released from impaired cells may reflect the cell status in testis.ObjectivesTo find if seminal exosomal microRNAs can reflect the Sertoli cell function in varicocoele.Materials and methodsExperimental left varicocoele rat model was established (n = 24), and patients with different grades of varicocoele (n = 104) were enrolled. Primary rat Sertoli cells were isolated with enzymatic hydrolysis. Exosomes were isolated from primary rat Sertoli cells, rat epididymis tissue, and human seminal plasma with polymer‐based precipitation method. Exosomal microRNAs were quantified with qPCR. Inhibin‐B was detected with enzyme immunoassay. The correlation analysis between microRNA and inhibin‐B was evaluated with Spearman's correlation.ResultsWe screened 12 previously reported hypoxia‐responsive microRNAs in the primary rat Sertoli cells and found that 4 exosomal microRNAs increased significantly in response to in vitro hypoxia treatment (P < .05). Of the 4 microRNAs, only miR‐210‐3p was upregulated in the rats with experimental varicocoele (P < .01). In the patients with varicocoele, we found that seminal exosomal miR‐210‐3p significantly increased in patients with grade II and III varicocoele (P < .01), and miR‐210‐3p negatively correlated with sperm count (P < .01) and seminal inhibin‐B expression (r = −0.39, P < .01). For the 30 patients with microsurgical varicocelectomy, the operation notably decreased miR‐210‐3p (P < .01).Discussion and ConclusionSeminal exosomal miR‐210‐3p may be a novel, sensitive, and non‐invasive biomarker of Sertoli cell damage in varicocoele.